Engineered in vivo nuclear fusion: application to regenerative medicine

描述（由申请人提供）：工程体内核聚变：在再生医学中的应用。本申请涉及广泛的挑战领域（11）再生医学和特定的挑战主题，11- dk -104：使用造血干细胞（HSC）再生或修复间质组织。该应用侧重于开发一种新的基于细胞的再生医学和基因治疗策略，该策略依赖于利用小鼠逆转录病毒包膜或人类病毒样融合基因产物，对造血干细胞或其他细胞群体进行基因工程改造，使其能够在体内移植后与器官和组织内的细胞有效融合。该方法寻求建立在最近对造血或其他干细胞转分化为意想不到的分化细胞的能力的研究基础上，这些研究表明，循环造血细胞和器官和组织内的细胞可以发生自发的细胞融合，特别是在组织损伤的反应中，并且可能代表器官和组织修复的重要机制。这项资助申请背后的假设是，基因技术可以提供一种显著提高移植细胞融合效率的方法，这种体内工程细胞-细胞融合可以为修复组织和提供有用的治疗方法提供广泛而强大的平台。这个为期两年的项目的一个主要目标是快速评估这项新技术的潜在广度，以便后续的研究能够立即集中在最有希望的治疗应用上。在一系列的体外实验中，我们将努力进一步了解通过融合性病毒包膜基因产物控制工程细胞-细胞融合的实验参数，重点是表征融合细胞的活力和命运，并确定融合性包膜或其相应受体的表达水平如何影响融合的绝对效率和融合核的平均数量。体内研究将集中于确定局部或全身递送不同类型供体细胞后可适应细胞融合的组织和器官范围。除了确定核移植到肌肉和其他组织和器官的效率外，还将确定来自不同细胞类型的供体核提供治疗性基因产物表达的能力，以及同种异体和异种细胞移植的免疫学后果。体内研究的一个特别重要的目标将是了解供体和受体细胞核之间的相互作用如何影响不同组织中供体和受体细胞核重编程和/或维持分化表型的程度。特别令人感兴趣的是，体内核融合是否可以用于将胰腺或肝脏中的细胞重编程为胰岛表型，以及/或通过将这些特化细胞的细胞核引入肌肉或其他组织，使特定细胞（如胰岛素或抗体分泌细胞）的特化分化功能得以保存。在通过造血干细胞移植系统地将细胞核传递到组织的努力中，一个重要的目标是直接确定与未转导细胞相比，融合基因产物的表达是否可以提供更高水平的供体来源细胞核的植入和再生修复。

DESCRIPTION (provided by applicant): Engineered in vivo nuclear fusion: application to regenerative medicine. This application addresses broad Challenge Area (11) Regenerative Medicine and specific Challenge Topic, 11-DK-104: Use of Hematopoietic Stem Cells (HSC) to regenerate or repair mesenchymal tissues. This application focuses on the development of a new cell-based strategy for regenerative medicine and gene therapy which depends upon the ability to genetically engineer hematopoietic stem cells or other cell populations to be capable of efficient fusion to cells within organs and tissues after their transplantation in vivo, using murine retroviral envelope or human viral-like fusogenic gene products. The approach seeks to build upon provocative recent studies of the ability of hematopoietic or other stem cells to transdifferentiate into unexpected differentiated cells which have suggested that spontaneous cell fusion involving circulating hematopoietic cells and cells within organs and tissues can occur, particularly in response to tissue injury, and may represent an important mechanism for the repair of organs and tissues. The hypothesis underlying this grant application is that genetic technologies can provide a means of dramatically increasing the efficiency of cell fusion of transplanted cells, and that such engineered cell-cell fusion in vivo can provide a broad and powerful platform for repairing tissues and for the delivery of useful therapeutics. A major goal of the two-year program is to rapidly assess the potential breadth of the new technology so that subsequent studies can immediately focus on the most promising therapeutic applications. In a series of in vitro experiments, effort will be made to further understand the experimental parameters governing engineered cell-cell fusion via fusogenic viral envelope gene products, with an emphasis on characterizing the viability and fate of fused cells, and determining how expression levels of either the fusogenic envelopes or their corresponding receptors affect the absolute efficiency of fusion and the average number of nuclei that are fused. In vivo studies will focus on a determination of the range of tissues and organs that are amenable to cell fusion after local or systemic delivery of different types of donor cells. In addition to determining the efficiency of nuclear transfer to muscle and other tissues and organs, the capacity of donor nuclei derived from different cell types to provide for the expression of therapeutic gene products, and the immunological consequences of the transplantation of allogeneic and xenogeneic cells, will be determined. A particularly important goal of the in vivo studies will be to understand how interactions between donor and recipient nuclei affect the extent of reprogramming and/or maintenance of the differentiated phenotype of the donor and recipient nuclei in different tissues. Of specific interest is whether in vivo nuclear fusion can be employed to reprogram cells in the pancreas or liver to the islet phenotype, and/or to enable the preservation of the specialized differentiated functions of specific cells, such as insulin or antibody secreting cells via introduction of nuclei from those specialized cells into muscle or other tissues. In efforts to systemically deliver nuclei to tissues via the transplantation of hematopoietic stem cells, an important objective is to directly determine whether the expression of fusogenic gene products can provide for increased levels of engraftment of donor derived nuclei and regenerative repair relative to untransduced cells. PUBLIC HEALTH RELEVANCE: The proposed research is highly relevant to human health, as the goal of the studies is to develop a novel cell transplantation approach to regenerative medicine. Such a therapy could have a major impact upon the treatment of many inherited and acquired diseases for which there are currently no effective treatments